Glycosylphosphatidylinositol-anchored proteins are required for the transport of detergent-resistant microdomain-associated membrane proteins Tat2p and Fur4p. (33/109)

In eukaryotic cells many cell surface proteins are attached to the membrane via the glycosylphosphatidylinositol (GPI) moiety. In yeast, GPI also plays important roles in the production of mannoprotein in the cell wall. We previously isolated gwt1 mutants and found that GWT1 is required for inositol acylation in the GPI biosynthetic pathway. In this study we isolated a new gwt1 mutant allele, gwt1-10, that shows not only high temperature sensitivity but also low temperature sensitivity. The gwt1-10 cells show impaired acyltransferase activity and attachment of GPI to proteins even at the permissive temperature. We identified TAT2, which encodes a high affinity tryptophan permease, as a multicopy suppressor of cold sensitivity in gwt1-10 cells. The gwt1-10 cells were also defective in the import of tryptophan, and a lack of tryptophan caused low temperature sensitivity. Microscopic observation revealed that Tat2p is not transported to the plasma membrane but is retained in the endoplasmic reticulum in gwt1-10 cells grown under tryptophan-poor conditions. We found that Tat2p was not associated with detergent-resistant membranes (DRMs), which are required for the recruitment of Tat2p to the plasma membrane. A similar result was obtained for Fur4p, a uracil permease localized in the DRMs of the plasma membrane. These results indicate that GPI-anchored proteins are required for the recruitment of membrane proteins Tat2p and Fur4p to the plasma membrane via DRMs, suggesting that some membrane proteins are redistributed in the cell in response to environmental and nutritional conditions due to an association with DRMs that is dependent on GPI-anchored proteins.  (+info)

Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease. (34/109)

The Ca2+/calmodulin-dependent phosphatase calcineurin promotes yeast survival during environmental stress. We identified Slm1 and Slm2 as calcineurin substrates required for sphingolipid-dependent processes. Slm1 and Slm2 bind to calcineurin via docking sites that are required for their dephosphorylation by calcineurin and are related to the PXIXIT motif identified in NFAT. In vivo, calcineurin mediates prolonged dephosphorylation of Slm1 and Slm2 during heat stress, and this response can be mimicked by exogenous addition of the sphingoid base phytosphingosine. Slm proteins also promote the growth of yeast cells in the presence of myriocin, an inhibitor of sphingolipid biosynthesis, and regulation of Slm proteins by calcineurin is required for their full activity under these conditions. During heat stress, sphingolipids signal turnover of the uracil permease, Fur4. In cells lacking Slm protein activity, stress-induced endocytosis of Fur4 is blocked, and Fur4 accumulates at the cell surface in a ubiquitinated form. Furthermore, cells expressing a version of Slm2 that cannot be dephosphorylated by calcineurin display an increased rate of Fur4 turnover during heat stress. Thus, calcineurin may modulate sphingolipid-dependent events through regulation of Slm1 and Slm2. These findings, in combination with previous work identifying Slm1 and Slm2 as targets of Mss4/phosphatidylinositol 4,5-bisphosphate and TORC2 signaling, suggest that Slm proteins integrate information from a variety of signaling pathways to coordinate the cellular response to heat stress.  (+info)

Tapping the nucleotide pool of the host: novel nucleotide carrier proteins of Protochlamydia amoebophila. (35/109)

Protochlamydia amoebophila UWE25 is related to the Chlamydiaceae comprising major pathogens of humans, but thrives as obligate intracellular symbiont in the protozoan host Acanthamoeba sp. The genome of P. amoebophila encodes five paralogous carrier proteins belonging to the nucleotide transporter (NTT) family. Here we report on three P. amoebophila NTT isoforms, PamNTT2, PamNTT3 and PamNTT5, which possess several conserved amino acid residues known to be critical for nucleotide transport. We demonstrated that these carrier proteins are able to transport nucleotides, although substrate specificities and mode of transport differ in an unexpected manner and are unique among known NTTs. PamNTT2 is a counter exchange transporter exhibiting submillimolar apparent affinities for all four RNA nucleotides, PamNTT3 catalyses an unidirectional proton-coupled transport confined to UTP, whereas PamNTT5 mediates a proton-energized GTP and ATP import. All NTT genes of P. amoebophila are transcribed during intracellular multiplication in acanthamoebae. The biochemical characterization of all five NTT proteins from P. amoebophila in this and previous studies uncovered that these metabolically impaired bacteria are intimately connected with their host cell's metabolism in a surprisingly complex manner.  (+info)

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family. (36/109)

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.  (+info)

Nucleotide-sugar transporters: structure, function and roles in vivo. (37/109)

The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.  (+info)

Isolation of new CHO cell mutants defective in CMP-sialic acid biosynthesis and transport. (38/109)

Sialic acid is a sugar typically found at the N-glycan termini of glycoproteins in mammalian cells. Lec3 CHO cell mutants are deficient in epimerase activity, due to a defect in the gene that encodes a bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sialic acid modification on the cell surface is partially affected in these cells. We have mutagenized Lec3 CHO cells and isolated six mutants (termed C2m) deficient in the cell surface expression of polysialic acid (PSA). Mutant C2m9 was partially defective in expression of cell-surface PSA and wheat germ agglutinin (WGA) binding, while in the other five mutants, both cell-surface PSA and WGA binding were undetectable. PSA expression was restored by complementation with the gene encoding the CMP-sialic acid transporter (CST), indicating that CST mutations were responsible for the phenotypes of the C2m cells. We characterized the CST mutations in these cells by Northern blotting and RT-PCR. C2m9 and C2m45 carried missense mutations resulting in glycine to glutamate substitutions at amino acids 217 (G217E) and 256 (G256E), respectively. C2m13, C2m39 and C2m31 had nonsense mutations that resulted in decreased CST mRNA stability, and C2m34 carried a putative splice site mutation. PSA and CD15s expression in CST-deficient Lec2 cells were partially rescued by G217E CST, but not by G256E CST, although both proteins were expressed at similar levels, and localized to the Golgi. These results indicate that the novel missense mutations isolated in this study affect CST activity.  (+info)

Two functionally divergent UDP-Gal nucleotide sugar transporters participate in phosphoglycan synthesis in Leishmania major. (39/109)

In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO(4)) repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Data base mining revealed 12 candidate NST genes in the L. major genome, including LPG2 as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L) and several pseudogenes. Gene knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although the single lpg5A(-) and lpg5B(-) mutants produced PGs, an lpg5A(-)/5B(-) double mutant was completely deficient. PG synthesis was restored in the lpg5A(-)/5B(-) mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, since the lpg5A(-) mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the lpg5B(-) mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally.  (+info)

The insulin-like growth factor-I-mTOR signaling pathway induces the mitochondrial pyrimidine nucleotide carrier to promote cell growth. (40/109)

The insulin/insulin-like growth factor (IGF) signaling pathway to mTOR is essential for the survival and growth of normal cells and also contributes to the genesis and progression of cancer. This signaling pathway is linked with regulation of mitochondrial function, but how is incompletely understood. Here we show that IGF-I and insulin induce rapid transcription of the mitochondrial pyrimidine nucleotide carrier PNC1, which shares significant identity with the essential yeast mitochondrial carrier Rim2p. PNC1 expression is dependent on PI-3 kinase and mTOR activity and is higher in transformed fibroblasts, cancer cell lines, and primary prostate cancers than in normal tissues. Overexpression of PNC1 enhances cell size, whereas suppression of PNC1 expression causes reduced cell size and retarded cell cycle progression and proliferation. Cells with reduced PNC1 expression have reduced mitochondrial UTP levels, but while mitochondrial membrane potential and cellular ATP are not altered, cellular ROS levels are increased. Overall the data indicate that PNC1 is a target of the IGF-I/mTOR pathway that is essential for mitochondrial activity in regulating cell growth and proliferation.  (+info)